Silyl lipids suitable for enhanced delivery of anti-viral therapeutics

ABSTRACT

This disclosure provides silyl lipid molecules in which one or more carbon-to-carbon double bonds in the lipophilic portion is substituted with a silicon atom. Guidance is provided by which the reader may make silyl lipid molecules from molecular building blocks, and then incorporate silyl lipid molecules into lipid nanoparticles (LNPs). The silyl LNPs can be used as carriers of pharmaceutical agents. Flexible steric and substitution patterns of silyl groups in the LNPs give the user a way to fine-tune physicochemical properties, achieving improved stability and clinical efficacy. This technology is useful for immunization or genetic therapy, such as the preparation and administration of RNA vaccines for protection against the virus that causes COVID-19.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.18/159,010, filed Jan. 24, 2023 (pending), which claims the prioritybenefit of U.S. provisional application 63/323,388, filed Mar. 24, 2022.The aforesaid priority applications are hereby incorporated herein byreference in their entireties for all purposes.

GOVERNMENT RIGHTS

This invention was made with government support under Grant No.1R03EB033487-01 awarded by the National Institutes of Health. The U.S.Government has certain rights in the invention.

FIELD OF THE INVENTION

The technology disclosed and claimed below relates generally to thefields of lipid chemistry, lipid microparticles, genetic therapy, andimmunopharmaceuticals. More specifically, it provides improvements inlipid nanoparticles with improved properties for use in vaccines.

BACKGROUND

COVID-19 is an ongoing global pandemic caused by a virus named as thesevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It wasfirst identified from an outbreak in December 2019. Attempts to containit at the initial site failed, allowing it to spread across the globe.

COVID-19 symptoms range from undetectable to deadly, but most commonlyinclude fever, dry cough, loss of a sense of smell, and fatigue. Severeillness is more likely in elderly patients and those with certainunderlying medical conditions. Infected persons are typically contagiousfor 10 days, and can spread the virus even if they do not developsymptoms. Mutations have produced many variants with different degreesof infectivity and virulence. As of January of 2023, COVID-19 has causedmore than 1.1 million deaths in the U.S. and 6.7 million deathsworldwide, making it one of the deadliest virus-induced diseases inhistory.

Vaccines against SARS-CoV-2 are widely credited for reducing theseverity and death caused by COVID-19. Some of the most rapidlydeveloped and successful vaccines are based on recombinant RNAtechnology: either viral vectors or messenger RNA (mRNA), which bothcause cells to express the spike protein presented on the surface ofSARS-CoV-2. This stimulates the immune system to develop protectiveantibody and T-cells against the spike protein.

The delivery of mRNA is achieved by a co-formulation of the moleculeinto lipid nanoparticles which protect the RNA strands and help theirabsorption into the cells. Krammer F (October 2020), Nature. 586 (7830):516-527; Kowalski PS et al. (April 2019) Molecular Therapy. 27 (4);Verbeke R et al, Nano Today. 28: 100766. Lipid nanoparticles for mRNAdelivery has been reviewed by X. Hou et al. (December 2021), NatureReview Materials 6:1078-1094.

RNA vaccines for COVID-19 that are currently authorized for clinical usein the U.S. and Europe have been developed and distributed byPfizer-BioNTech and by Moderna. They play central roles in theclinician's arsenal to decrease spread of COVID-19. Current RNA lipidvaccines must be stored at −20° C. or −80° C. Severe allergic reactionsare rare. The COVID-19 RNA vaccine from CureVac failed in clinicaltrials (Press release, Jun. 30, 2021). Lipid technologies incorporatedinto the Moderna and BioNTech vaccines are described in U.S. Pat. Nos.10,266,485 and 10,576,146 respectively.

SUMMARY

This disclosure provides silyl lipids in which one or morecarbon-to-carbon double bonds in the lipophilic portion is substitutedwith a silicon atom. Guidance is provided by which the reader may makesilyl lipids from molecular building blocks, and then incorporate silyllipids into lipid nanoparticles (LNPs). The silyl LNPs can be formulatedas carriers of pharmaceutical agents. Flexible steric and substitutionpatterns of silyl groups in the LNPs allow the user to fine-tunephysicochemical properties, achieving improved stability and clinicalefficacy. This technology is useful for immunization or genetic therapy,such as the preparation and administration of RNA vaccines forprotection against the virus that causes COVID-19.

Included as part of this disclosure are silyl lipids, and nanoparticlescontaining such silyl lipids. The silyl lipids described in more detailin the sections that follow include lipids that have the structure shownin Formula I:

P[[-R ⁶ —Si(R ^(4a))(R ^(4b))- ]_(n6)-R ⁷]_(n0)

Formula I

P is a polar headgroup containing 2 to 25 carbon atoms, 2 to 8 oxygenatoms, and at least one nitrogen atom that is ionizable or positivelycharged; R^(4a) and R^(4b) are independently selected from methyl and alinear alkyl group of at least 4 carbon atoms. R⁶ and R⁷ arehydrocarbons of 2 to 20, 2 to 15, or 2 to 12 carbon atoms: usuallylinear, but optionally branched, and optionally having a different endgroup R⁸, which may be a substituted or unsubstituted hydrocarbon. TheR⁷ and R⁸ for each of the tails may be the same or different. n6 (thenumber of silicon atoms per lipid tail) is between 1 and 4; and n0 (thenumber of lipid tails per lipid molecule) is between 1 and 6.

R^(4a) and R^(4b) may both be methyl. The lipids may have branched fattyacid tails wherein at least one of R^(4a) and R^(4b) is a linear alkylgroup of at least 4 carbon atoms. The silyl lipids may contain two[—(CH₂)_(n2)—Si(R^(4a))(R^(4b))-(CH₂)_(n3)—CH₃] groups which areidentical or non-identical. The polar headgroup of the silyl lipids maycontains or be conjugated to polyethylene glycol.

The silyl lipids may have the structure shown in Formula III:

R¹, R², and R³ are independently H, —CH₃, or a substituted orunsubstituted hydrocarbon containing two to ten carbon atoms, X¹ isnitrogen or —(OPO₃)—, X² and X³ are independently methylene or carbonylor together are a quaternary carbon; R⁴ and R⁵ are independentlyselected from methyl and a linear or non-linear (optionally substituted)hydrocarbon of 4 to 12 carbon atoms; n1=1 to 6; and n2, n3, n4 and n5are independently 4 to 12.

X¹ may be nitrogen, or it may be phosphate. R¹, R², and R³ are all —CH₃.In one example. R¹ and R² are both —CH₃ and R³ is a substitutedhydrocarbon containing two to ten carbon atoms. In another example, R¹is H, R² is —CH₃ and R³ is a substituted or unsubstituted, optionallylinear hydrocarbon containing two to ten carbon atoms.

Referring to Formula III, any one or more of the following features maybe included in any combination: n1 is 1; X² and X³ are both methylene orboth carbonyl; n2 and n4 are both 7; n3 and n5 are both 7; R⁴ and R⁵ areboth methyl; and/or R⁴ and R⁵ are both independently a linearhydrocarbon of 6 to 10 carbon atoms; R^(8a) and R^(8b) are each methylor a saturated or unsaturated hydrocarbon of 2 to 10 or 2 to 6 carbons,such as —CH₃, cyclohexyl, or phenyl. Optionally, some of the silyllipids in the preparation are PEGylated, whereby any one or more of R¹,R², and R³ contain —O—[CH₂—CH₂—O- ]_(n9)-R⁹. The R⁹ substituent may be—OH, —OCH₃, —NH₂, or a substituted hydrocarbon, and n9 is an integer of10 or more.

A variant of Formula III incorporating a silicon substituted carbocycle,such as silicon substituted cyclobutane, cyclopentane, or cyclohexane.This is depicted in Formula IIIa, where n is 0 to 4, typically 1 or 2.

Nanoparticles made from the silyl lipids disclosed herein typicallycontain at least 2%, 5%, or 10% silyl lipids. In certain implementationsof the technology, at least 25% of lipids in the nanoparticles are silyllipids, and/or at least 50% of the silyl lipids in the nanoparticles arecationic and/or comprise a headgroup that is ionizable. The lipidnanoparticle may contain cationic lipids, ionizable lipids, andPEGylated lipids all together, any one, two, or all three of which maybe silylated, or contain a proportion that is in silylated form. Thelipid nanoparticle may also contain cholesterol.

The lipid nanoparticles may be solid nanoparticles comprising a lipidmonolayer surrounding a drug payload. Alternatively, they may beliposomes enveloping a drug payload. The solid nanoparticles orliposomes may have an median diameter between 20 and 500 nm, or haveother dimensions as put forth below.

The drug payload in the nanoparticles may comprise a nucleic acid forgene therapy or immunization of a subject in need thereof. By way ofexample, the drug payload comprises a messenger RNA (mRNA) or othernucleic acid that encodes a tumor antigen for eliciting an immunologicalresponse against a tumor in the subject being treated. Alternatively,the payload may include a protein component of a pathogen or a nucleicacid encoding such protein component, such as the spike protein ofSARS-CoV-2 (the virus that causes COVID-19) or an immunogenic portionthereof.

This disclosure includes methods of gene therapy and methods ofeliciting a specific immune response. A pharmaceutical productcomprising a plurality of nanoparticles as put forth herein isadministered to a subject in need thereof. The disclosure includes theuse of the silyl lipids and nanoparticles as heretofore described fortreating or preventing a disease or eliciting an immune response in asubject, or for preparation of a medicament for such purpose.

This disclosure also provides a method of improving a previous design orpreparation of lipid nanoparticles. To do this, the user determines oneor more lipids contained in the previous preparation that have one ormore cis-unsaturated carbon-carbon bonds in one or more fatty acids inthe lipid(s). They then design a silyl lipid in which one or more of thecis-unsaturated carbon-carbon bonds is substituted, for example, with[-CH₂—Si ((CH)₃)₂—CH₂-], or with [-CH₂—Si(R^(x))₂-CH₂-], wherein eachR^(x) is independently a hydrocarbon. The user then produces an improvedpreparation of lipid nanoparticles that contain the silyl lipid as wellas or in place of one or more of the lipids contained in the previouspreparation.

The silyl lipid may have one or more cis-unsaturated carbon-carbon bondthat have been silylated. Examples include but are not limited to DOTMA,DOTAP, DOSPA, ePC, DLin-MC3-DMA, A2,iso-5-2DC18, OF-Deg-Lin, and DOPE.

Without intending to otherwise limit the practice of the technology putforth in this disclosure, an example is use of the technology to preparean immunogenic composition formulated for administration to a subjectfor prevention or treatment of COVID-19. The composition includes aplurality of nanoparticles that contains a drug payload. Typically, atleast 2%, 5%, 10%, or 20%, or 50% of lipids in the nanoparticles aresilyl lipids having the structure shown in Formula I, II, III, or IIIa,and including any one or more of the features put forth below in anycombination.

To elicit the immune response in the subject being treated, the drugpayload includes one or more protein components of SARS-CoV-2 (the virusthat causes COVID-19), optionally in combination with an immunologicaladjuvant, and/or one or more nucleic acids encoding such proteincomponents. For example, the drug payload may include a messenger RNA(mRNA) encoding any one or more of the S (spike), E (envelope), M(membrane), and/or N (nucleocapsid) of the SARS-CoV-2 virus (includingany naturally occurring or artificial variants thereof), or andimmunogenic portions thereof in any combination.

The reference sequence for the severe acute respiratory syndromecoronavirus 2 isolate Wuhan-Hu-1 can be obtained from GenBank accessionNo. NC_045512 (Wu F et al., Nature 579 (7798): 265-269, 2020). The aminoacid sequence of protein components of SARS-CoV-2 virus can be obtainedfrom GenBank accession No. MT108784 (Thi Nhu Thao, T., Nature 582(7813), 561-565, 2020). For example, silyl lipid nanoparticle vaccinesaccording to this disclosure can be generated with a drug payload thatcomprises a nucleic acid or mRNA that encodes a protein that is at least50%, 70%, 80%, 90%, or 95% identical to the spike protein sequencelisted in GenBank accession No. MT 108784, deposited on 11 Feb. 2022, orany immunogenic portion thereof.

The silyl lipids and the lipid nanoparticle may have any of the featuresreferred to above. In a particular formulation, the lipids in thenanoparticle include cationic lipids, ionizable lipids, and PEGylatedlipids together in any effective amount and proportion, any one or moreof which are silyl lipids. For example, the nanoparticle may contain anyone or more of the following components in silylated form: DOTMA(1,2-di-O-octadecenyl-3-trimethylammonium-propane), DOTAP(N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride), DOPE(2-dioleoyl-sn-glycero-3-phosphoethanolamine), and PEGylated DOPE.

This disclosure includes methods for inducing a specific immune responsein a subject at risk of contracting COVID-19 by administering to thesubject the nanoparticles referred to above. Other possible drugpayloads and their use are put forward in Tables 1 and 2 below.

Aspects and embodiments of the technology are presented in thedescription, drawings, and claims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show an example in which a silicon atom in the form of asilyldimethyl or silylcycloakyl group are incorporated into the targetlipid DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium-propane) in placeof a carbon-carbon double bond. Lipid nanoparticles (LNPs) manufacturedwith silyl lipids have improved physicochemical and pharmaceuticalproperties compared with their non-silylated counterparts.

FIG. 2 is a scheme of chemical reagents and synthesis, whereby severalfamilies of molecular building blocks and precursors are used to producesilyl lipids with a silicon atom placed at a precise location in thelipid structure.

FIGS. 3A and 3B show lipids with unsaturated fatty acid tails that arecurrently used or proposed for use in LNPs. They can be converted totheir silyl analogs and used to prepare improved LNPs in accordance withthis disclosure.

FIG. 4 shows DOPE (2-dioleoyl-sn-glycero-3-phosphoethanolamine) in itsstandard form, and as a silyl lipid incorporating a single methylatedsilicon atom in the form of [—Si(CH₂)₂-] into each of the two lipidtails. Also shown are PEGylated forms of both DOPE and Silyl-DOPE,wherein the charged head group is conjugated to polyethylene glycol.Nanoparticles containing cationic lipids, ionizable lipids, andPEGylated lipids all together as part of the lipid content (at least oneof which is in silylated form) have beneficial properties that improvepreparation, stability, and therapeutic efficacy.

FIGS. 5A to 5C illustrate effects of lipid structure on properties ofliposomes made from such lipids. There was a substantial effect onyield, particle diameter, and zeta potential.

FIG. 6 shows possible alternative lipophilic groups placed at or nearthe lipid tail of silyl lipids to instill nanoparticles made therefromwith beneficial properties.

FIGS. 7A to 7C illustrate how structure structural features such as taillength, positioning of the silicon group, and branching can affectproperties of nanoparticles.

DETAILED DESCRIPTION

Before the making of the technology provided below, lipid nanoparticles(LNPs) for drug delivery have been designed by (1) modifying theprocedure for forming nanoparticles using ionizable cationic lipids, and(2) modifying the chemistry of the hydrophobic head-group. There hasbeen less emphasis on structural modifications of the lipid component,due to a general limitation in lipid pharmacophore options.

This disclosure provides new silyl-containing lipids that can be used toprepare improved LNPs with superior properties: better stability,powerful adjuvant potential, and enhanced delivery activity. The familyof silyl-lipids provided in this disclosure can be fine-tuned to createnew structures, varying the position of the silyl group, incorporatingbranched structures, and varying chain length.

Membrane fluidity is a contributing factor in transfection and thesubsequent endosomal release of the drug payload after endocytosis. Thepresence of structural features such as a cis-unsaturation in the lipidtail helps prevent tight membrane packing. This promotes membranefluidity, and positively influences transfection into cells. Thedimethyl silyl or the silacycloalkane group has steric effects that canmimic a cis-alkene in bond distances and angles, potentially enhancingmembrane fluidity. Moving the position of the silyl group and installingvarious substitutions on the silyl group, including aryl, alkyl, andcycloalkyl can also be used to fine-tune membrane fluidity and otherproperties of nanoparticles that contain them.

The silicon group can comprise two methyls or other R groups, or asilicon substituted cycloalkyl group such as silalyclobutyl,silacyclopentyl or silylcyclohexyl. Besides effects on membranefluidity, replacing cis-alkene groups in non-saturated lipids with suchsilicon groups helps stabilize nanoparticles, rendering them less proneto oxidative damage.

Use of Silyl Lipids for Preparing LNPs

Liposomes such as nucleic acid complexed lipid nanoparticles (LNPs) havebeen successfully used in drug delivery and formulation of sensitiveRNA-based therapeutics, including the recent example of the mRNASar-COV-2 vaccine. This disclosure details the synthesis and evaluationof novel silyl-containing lipid structures to access innovative cationiclipid structures representing new chemical space for biomedicalresearch.

FIGS. 1A and 1B show an example in which a silicon atom in the form of asilyldimethyl or silylcycloakyl group are incorporated into the targetlipid DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium-propane) in placeof a carbon-carbon double bond. The position of the silicon atom on thelipid tail, the option of branching the tail at the silicon atom, andthe ability to use different headgroups represents a modular design bywhich the user may optimize lipid properties for a chosen purpose. SilylLNPs containing, for example, a therapeutic RNA molecule has highertransfection efficacy, wide cell tolerability, and a longer shelf life.

FIG. 2 is a scheme of chemical reagents and synthesis, whereby severalfamilies of molecular building blocks and precursors are used to producesilyl lipids with a silicon atom placed at a precise location in thelipid structure. Different metal catalysts can be used forhydrosilylation reactions, where the catalyst is selected based on theyield and isomeric purity of the silylated lipid product. The synthesisalso has flexibility since the hydrosilylation step can be performed ina different order to access the lipid structures.

Three classes of cationic silyl-lipids may be synthesized in modularfashion to access structural and conformational cationic lipid analogsthat have implications in the phase transition temperature and thefluidity of the bilayer, influencing the stability, toxicity, andfusogenicity of silyl-LNPs. This technology is focused on the synthesisof novel silyl-containing lipids as diverse cationic lipid vectors usingcatalytic hydrosilylation methods, particularly focusing on the modularincorporation of a silyl dimethyl group as a bioisostere of a ciscarbon-carbon double bond of known unsaturated cationic lipid vectors,as well as other silyl groups with relevant properties to modulate thebranching and chain length of the resulting lipid. Target moleculesinclude silyl analogs of DOTMA, DOTAP (N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride) and DOSPA (2′-(1″,2″-dioleoyloxypropyldimethyl-ammonium bromide)-N-ethyl-6-amidosperminetetratrifluoroacetic acid).

Use of Silyl-Lipid Containing LNPs for Drug Delivery

Unsaturated and branching fatty acid chains have shown to provide afluidity to the bilayer by disrupting membrane packing, facilitatingendosomal escape and oligonucleotide delivery. Cationic and ionizablelipid vectors, including DOTMA, DOTAP, DOSPA and DLin-MC3-DMA lipids,are examples of optimized cis-unsaturated lipids for improvedtransfection efficiency but a disadvantage to incorporating unsaturationis cis-trans isomerization of alkenes and susceptibility to oxidation,leading to low stability in storage and compatibility with diverse celltypes.

Silanes and siloxanes have been incorporated into synthesized productsin other fields, such as materials and inorganic chemistry. Thisdisclosure shows that unique properties of silicon can be exploited forbiomedical applications. The flexible steric and substitution patternsof silyl groups allow tunable reactivity, stability, and solubility.

Some of the properties relevant for medicinal and clinical applicationsare the following:

-   -   1. The C—Si bond is stable under physiological conditions;    -   2. There is no known inherent “element-specific” toxicity of        silicon containing compounds;    -   3. The silicon atom has a larger covalent radius with 20% longer        Si—X bonds, compared with C—X bonds, and provides higher        conformational flexibility;    -   4. The electropositive nature and bond-polarization of silicon        (relative to C, N, O) contributes to an electron-deficient        center;    -   5. Trialkylsilyl groups are more lipophilic than the        corresponding trialkylmethyl groups (LogP for        trimethylsilyl-benzene=4.7 vs LogP for t-butylbenzene=4.0); and    -   6. Silicon can prevent or alter oxidative metabolism and        metabolic fate to avoid toxic metabolites.

Structure of Silyl Lipids

Silyl lipids of this disclosure typically are amphipathic, having acharged headgroup, and one, two, or more than two lipophilic tails. Thesilicon atoms are located in the tails, taking the place of one or morecis alkene groups of previously known unsaturated fatty acids, orotherwise imparting solubility characteristics in a lipid monolayer orbilayer of interest to the user.

Particular silyl lipids in this class may be depicted as shown inFormula I:

P[[-R ⁶ —Si(R ^(4a))(R ^(4b))-]_(n6)-R ⁷]_(n0)

Formula I

When R⁶ and R⁷ are saturated allylene and alkyl groups respectively,this may be written as shown in Formula II:

P[[—(CH ₂)_(n2) —Si(R ^(4a))(R^(4b))-]_(n6)-(CH ₂)_(n3)-R ⁸]_(n0)

Formula II

P is a polar headgroup, typically containing 2 to 25 or more carbonatoms and 2 to 8 or more oxygen atoms. It is a hydrocarbon that issaturated or unsaturated and substituted or unsubstituted, andoptionally contains a cyclic hydrocarbon or aromatic substituent. It mayalso contain least one nitrogen atom that is ionizable or positivelycharged; and/or a phosphate (═OPO₃). When used to form lipidnanoparticles, R^(4a) and R^(4b) are usually selected from methyl and alinear alkyl group of at least 4 carbon atoms.

n6 is the number of silicon atoms per lipid tail. It is typically aninteger between 1 and 4. If n6>1, then n2, R_(4a), and R_(4b) in eachsection of the tail may be the same or different. n0 is the number oflipid tails per lipid molecule, typically an integer between 1 and 8. InFormula I, R⁶ and R⁷ are an alkylene or alkyl group respectively of 2 to12 carbon atoms, typically linear but optionally branched, typicallysaturated but optionally unsaturated. Optionally, each of the tails mayindependently have a non-linear end group R⁸, which may be a substitutedor unsubstituted hydrocarbon. For each of the lipid tails, R⁷ in FormulaI and R⁸ in Formula II may be the same or different. n6 (the number ofsilicon atoms per lipid tail) is between 1 and 4; and n0 (the number oflipid tails per lipid molecule) is between 1 and 6. In both Formula Iand Formula II, R^(4a) and R^(4b) together may form a siliconsubstituted cyclical hydrocarbon, for example, in the form—Si(═(CH₂)n)-, where n is between 2 and 6, typically 3 or 4.

In Formula II, n2 and n3 (the number of methylene groups surroundingeach silicon atom) are typically from 2 to 12. R^(4a) and R^(4b) mayboth be methyl, in which case the fatty acid forms a single chain withan internal —CH₂—Si(CH₃)₂—CH₂-. Alternatively, one or more of the lipidsmay have branched fatty acid tails, wherein at least one of R^(4a) andR^(4b) is a linear saturated or unsaturated hydrocarbon of at least 2,4, or 8 carbon atoms. If n0>1, the lipid tails may be identical ordifferent.

Some lipids useful for making LNPs have the structure shown in FormulaIII:

The group (R¹,R²,R³)—represents the charged headgroup, which is linkedin this example to a glycerol or acyl glycerol bearing two fatty acids.In this example, R¹, R², and R³ are independently H, —CH₃, or asubstituted or unsubstituted hydrocarbon containing two to ten carbonatoms or more, optionally containing cyclical or aromatic substituents.Typically X¹ is nitrogen, but it may also be phosphate, which case thesilyl lipid is a phospholipid.

The headgroup (being the hydrophilic portion) may be charged oruncharged. Lipids that are permanently cationic can be formed, forexample, by making X¹ a quaternary nitrogen, wherein each R¹, R², and R³contains at least one carbon atom: for example, the silyl equivalent of1,2-di-O-octadecenyl-3-trimethylammonium-propane (DOTMA), or1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). If at least one of R¹,R², and R³ is hydrogen, the molecule will be ionizable, which means thatthe lipid will be protonated at low or moderate pH to a positive form.In some instances, ionizable lipids are beneficial for mRNA delivery invivo, because neutral lipids have less interactions with anionicmembranes of blood cells, thereby improving biocompatibility.

If X¹ is phosphate, a negatively charged substituent is included to theheadgroup. If this is the only charged substituent, then the lipid willbe negatively charged or anionic at neutral pH. The lipid may containboth a phosphate at position and an ionizable nitrogen as part of R¹,R², or R³. In this case, the headgroup and the lipid will be at leastzwitterionic, being neutral, positive, or negatively charged dependingon other ionizable groups that may also be present as part of theheadgroup structure. An example is a silylated form of2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), namely Silyl-DOPE,shown in FIG. 4 .

As depicted in Formula III, X² and X³ are independently methylene orcarboxyl, or together constitute a quaternary carbon. R⁴ and R⁵ areindependently selected from methyl and a linear alkyl group of 4 to 12carbon atoms. n1 (the number of carbon atoms in the link between theheadgroup and the fatty acid portion) is often 1 or between 1 and 4 or 1and 8, inclusive. With respect to each of the lipophilic tails—that isto say, [[-R⁶—Si(R^(4a))(R^(4b))-]_(n6)-R⁷] in Formula I,[[—(CH₂)_(n2)—Si(R^(4a))(R^(4b))-]_(n6)—(CH₃ or -R⁸] in Formula II, or[—(CH₂)_(n2)—Si(R⁴)(CH₃)—(CH₂)_(n3)—CH₃ or -R^(8a)] and[—(CH₂)_(n4)—Si(R⁵)(CH₃)—(CH₂)_(n5)—CH₃ or R^(8b)] in Formula III orIIIa—n2, n3, n4 and n5 are independently 4 to 12. R^(8a) and R^(8b) areindependently methyl or a saturated or unsaturated hydrocarbon of 2 to10 carbon atoms.

By way of illustration, R¹, R², and R³ in Formula III may all be —CH₃,or R¹ and R² are both —CH₃ while R³ is an alkyl or heteroalkyl groupcontaining two to ten carbon atoms. In Formula III, X² and X³ may beindependently methylene or carbonyl. In some of the examples shown inthe drawings, n2 and n4 are both 7, and n3 and n5 are both 7. Thesilicon atom in each fatty acid may be blocked, herein R⁴ and R⁵ areboth methyl.

Alternatively, each of the tails may branch at the silicon atom into asecond tail, wherein one or other or both of R⁴ and R⁵ are independentlya linear alkyl group of 4 to 12 or 6 to 10 carbon atoms.

An included is a variant of Formula III incorporating a siliconsubstituted carbocycle, such as silicon substituted cyclobutane,cyclopentane, or cyclohexane. This is depicted in Formula IIIa, where nis 0 to 4, typically 1 or 2.

FIG. 4 depicts PEGylated forms of both DOPE and Silyl-DOPE. Included aspart of this disclosure are PEGylated forms of any of the silyl lipidsput forth herein. A “PEGylated” lipid is a lipid that has a polyethyleneglycol substituent contained in or covalently lined to the head group.

Referring to Formulas III and IIIa, any one or more of R¹, R², and R³contain —O—[CH₂—CH₂—O- ]_(n9)-R⁹. The substituent R⁹ is —OH, —OCH₃,—NH₂, or a substituted hydrocarbon (usually charged, ionizable, orotherwise polar). n9 is an integer of 10 or more, typically between 10and 1000, rendering silyl lipids that are between approximately 500 Daand 40,000 Da in weight. Also contemplated are PEGylated silyl lipidscontaining in their headgroups a polyethylene glycol or equivalentthereof that is branched, Y shaped or comb shaped. Silyl lipids have lowdispersion and are typically amphipathic (soluble in both polar andnon-polar solvents). They are generally non-toxic and biodegradable.

Procedures for LNP Preparation

Any suitable process to prepare LNP may be used, such as high shearhomogenization and ultrasound, solvent emulsification/evaporation, ormicroemulsion. General methods for preparing nanoparticles aredescribed, for example, in U.S. Pat. Nos. 9,393,215 (Glaxo Smith-Klein)and 10,266,485 (Moderna). The nanoprecipitation method and solventdisplacement method are described in U.S. Pat. No. 5,049,322.

A polymer is dissolved in an organic solvent such as acetone or ethanol.The resulting organic solution is combined with a further solvent, whichis miscible with the organic solvent while being a non-solvent for thepolymer, typically an aqueous solution such as deionized water, normalsaline, or a buffered solution. A surfactant may also be present.

The organic solution and aqueous solutions are then combined in suitablerelative volumes. For example, the organic solution may be poured orinjected into the non-solvent while stirring, or vice versa. Byselecting a system in which the polymer is soluble in the organicsolvent, while being significantly less soluble in the miscible blend ofthe organic solvent with the non-solvent, a suspension of nanoparticlesmay be formed virtually instantaneously. Subsequently, the organicsolvent can be eliminated from the suspension, for example, byevaporation under ambient conditions or evaporation under reducedpressure and/or elevated temperature. Pharmaceutical agents may be addedto the organic solution, if in oil-soluble or oil-dispersible form or tothe aqueous solution, if in water-soluble or water-dispersible form.

Surfactants useful for making lipid nanoparticles include variousdetergents, dispersing agents, suspending agents, and emulsionstabilizers. The amount of surfactant will be effective to promoteacceptable nanoparticle suspension (and resuspension afterlyophilization).

Cryoprotective agents can be added to the compositions to preventnanoparticle agglomeration from occurring when lyophilized compositionsin accordance with the invention are resuspended. Depending on the LNPformulation, suitable cryoprotective agents may include amino acids suchas glutamic acid and arginine; polyols, including diols such as ethyleneglycol, and/or carbohydrates (monosaccharides, disaccharides,polysaccharides such as cellobiose; and alditols such as xylitol andsorbitol.

Features of Lipid Nanoparticles Containing Silyl Lipids

A lipid nanoparticle may be a lipophilic or amphipathic core containinga drug payload, coated with a single lipid layer. Alternatively, it maybe a liposome or other multilamellar particle comprising a lipid bilayerwith the drug payload enveloped inside.

The LNP may contain any percentage of silyl lipids that is useful. LNPsmay be characterized in terms of the percentage of lipids in apreparation that are silyl lipids, and the percentage of silyl lipidsthat have a particular property. Unless specified otherwise, a silyl LNPpreparation of this disclosure will have a lipid composition in which atleast 5% of the lipids are silyl lipids, and at least 5% of the silyllipids are ionizable or cationic (percent per mole). Other useful rangesare LNPs wherein at least 2%, 10%, 20%, or 50%, or between 1% and 50% or5% to 30% of the lipid molecules are silyl lipids; and at least 10%,20%, or 50%, or between 1% and 50% or 5% to 30% of the silyl lipidmolecules are ionizable and/or cationic (percent per mole).

In addition to cationic or ionizable lipids, LNP formulations maycontain other lipid components. Lipids for optional inclusion includetriglycerides, diglycerides, monoglycerides, fatty acids, steroids, andwaxes. The non-silyl lipids may be saturated or unsaturated. Includedare naturally occurring or artificial phospholipids (for example,phosphatidylcholine and phosphatidylethanolamine), and polyethyleneglycol (PEG)-functionalized lipids. These may be included, for example,to improve nanoparticle properties, such as particle stability, deliveryefficacy, tolerability and biodistribution. Alternatively or inaddition, a sterol such as cholesterol or a derivative thereof may beincluded in the LNP preparation to enhance particle stability bymodulating membrane integrity and rigidity. Exemplary inclusions in LNPsof this disclosure are artificial lipids that are known to createpharmaceutically valuable LNPs, such as DOTMA, DOPE, DOTAP, or any ofthe lipids described in the article by X. Hou et al., Nat Rev Materials2021, 10:1-17, or in U.S. Pat. Nos. 9,393,215 and 10,266,485 that arenot silylated. The lipid balance in the LNP preparation is chosen toachieve satisfactory delivery efficacy and biodistribution of thepharmaceutical agent.

Included as part of this disclosure are lipid nanoparticles thatcomprise three types of lipid components together—each serving a uniquerole in transfection and delivery. Any one or two of these, or all threeof which may include a proportion that is silylated. The three-componentformulation can be used to enhance transfection and delivery of thelipid nanoparticles and their cargoes. The three components are cationiclipids (such as DOTMA and DOTAP), ionizable lipids (such as DOPE) andPEGylated lipids. Cholesterol is optionally included as a forthcomponent.

Cationic lipids have a permanent positive charge and facilitate in theencapsulation of RNA during lipid nanoparticle formation. Ionizablelipids can be protonated or deprotonated at the amine position dependingon pH. These ionizable lipids facilitate in endosomal escape of the RNAby becoming protonated in the endosome after cellular uptake andpromoting the Hexagonal II phase conformation. PEGylated lipids mayincrease the structural stability of nanoparticles made therefrom,improving blood circulation time and biodistribution of thenanoparticles. Cholesterol is used to maintain the structural stabilityof the lipid nanoparticles.

An LNP designed and manufactured in accordance with this technology istypically spherical with an average diameter between 10 and 1000nanometers. Working size range will depend on the method of manufacture,and the intended stability and clinical effect. Working ranges for solidnanoparticles or liposomes are, for example, at least 10, 20, 50, 100,or 200 nm in diameter, no more than 50, 100, 200, or 500 nm in diameter,or between 20 and 500 nm, 10 and 200 nm, or 100 and 500 nm.

The choice of a particular silyl lipid used, its structure, theproportion of silyl lipids to non-silyl lipids, and other featurespotentially affect the properties of nanoparticles made therefrom: forexample, yield, particle size, zeta potential, membrane fusion,deliverability of a drug payload, and effectiveness as a deliveryvehicle in vivo. All these variables can be optimized by empiricaltesting.

For colloidal dispersions, zeta potential is a useful measurement of thedifference in electrokinetic potential between the dispersion medium andthe stationary layer of fluid attached to the dispersed particle. Themagnitude of the zeta potential indicates the degree of electrostaticrepulsion between adjacent, similarly charged particles in a dispersion.A high zeta potential is an indicator of stability (resistance toaggregation). Both zeta potential and size can be determined using azeta Potential Analyzer. Clogston JD et al., Methods Mol Biol.2011;697:63-70.

FIGS. 5A to 5C and TABLE 1A provide an illustration of such empiricaldetermination: the effect of the length of the lipid tail, the endgroup, and the positioning of the silyl group. In this illustration,other properties of the lipid structure and liposome composition werekept constant. The liposome formulations just contained the cationicsilyl lipids indicated, and Milli Q™ purified water.

TABLE 1A Cationic silyl lipid structures and the effect on nanoparticlesproduced Yield Avg. Size Avg. zeta Preparation n1 n2 R (%) (nm)Potential (mV) QP5SiCy 2 0 Cyclohexyl 42 80  71; 124 QP5SiPh 2 0 Phenyl39 80 62 QP5Si4 2 3 CH₃ 52 21 65 QP5Si8 2 7 CH₃ 25 38  69; 112 QP5Si11 210 CH₃ 22 59 60 QP8Si8 5 7 CH₃ 7 38 67 QP8Si6 5 5 CH₃ 6 38 62 QP8Si4 5 3CH₃ 9 24 33; 44 QP7Si9 4 8 CH₃ 4 59 74 QP8SiCy 5 0 Cyclohexyl 48 29 57;71 QP8SiPh 5 0 Phenyl 46 21 59; 73

FIG. 5A and the first four columns of TABLE 1A show the choices made asto the structure of the lipid. FIGS. 5B and 5C graph the results. Therewas a substantial effect of these input parameters on yield, particlediameter, and zeta potential.

In some circumstances, putting a more bulky hydrophobic group such ascyclohexyl and phenyl at the end of the lipid tail may have thefollowing benefits:

(1) decrease the length and hydrodynamic diameter of the silyl-LNPsresulting in a smaller LNP for RNA delivery that is more able to getinside the cell via endocytosis, and (2) disrupt lipid tail packing andpromoting endosomal escape of RNA inside the cell resulting in increasedtransfection efficiency. The cyclohexyl or phenyl group may also help tofine tune interactions between lipid tails that are relevant forfluidity, such as potential pi-pi stacking interactions of the phenylrings.

FIG. 6 shows some examples of possible alternatives to —CH₃ placed at ornear the lipid tail of silyl lipids to instill nanoparticles madetherefrom with such advantages.

FIGS. 7A to 7C and TABLE 1B provide another illustration of howstructure of the silyl lipid can affect properties of nanoparticles.

TABLE 1B Cationic silyl lipid structures and the effect on nanoparticlesproduced Avg size Avg. zeta n1 n2 R (nm) potential (mV) 6,Si,8-DOTMA 5 7CH₃ 216 64 8,Si,8-DOTMA 7 7 CH₃ 206 56; 74 8,Si,(8)₃-DOTMA 7 7 CH₃ 18464 8,Si,Ph-DOTMA 7 0 CH₃ 215 62 6,Si,4-DOTMA 5 3 Phenyl 79 6510,Si,6-DOTMA 9 5 CH₃ 65 52 (DOTMA)/cholesterol liposome size: 126 nm(DOTMA)/cholesterol zeta potential: 51 mV

Six synthesized sila-DOTMA analogs were found to be capable of formingliposomes with similar size and zeta potential compared with nativeDOTMA. Structural features such as tail length, positioning of thesilicon group (n1, n2), and branching (as in 8,Si,(8)₃-DOTMA) caninfluence liposome properties.

Adaptation of Previous LNP Technology Using Silyl Lipids

To implement this technology in other ways, the user is not constrainedto particular formulas, structures, or named compounds referred to inthis disclosure. They may be guided instead by empirically adjusting oroptimizing the structure in silico and/or by experimentation in vitro.

FIGS. 3A and 3B show lipids that have been developed or shown to beeffective in imparting LNPs with particular properties. Any known lipidor lipid-like structure can be used as a starting model for thedevelopment of silyl lipids and silyl LNPs. For example, a siliconlinkage in the form of [—CH₂≤Si(R^(4a))(R^(4b))—CH₂-],[—(CH₂)—Si(CH₂)(R⁴)—CH₂-], [—(CH₂)—Si(CH₂)_(S)—CH₂-], or—CH₂—Si(—CH₂-(CH₂)_(n)—CH₂-)—CH₂- (where n is 0 to 4) may be insertedinto a fatty acid tail or linear alkyl group (saturated or unsaturated)at any place that is desired.

One approach to improve a previously designed or characterizedpreparation of lipid nanoparticles is to determine lipids contained inthe previous preparation that have one or more cis-unsaturatedcarbon-carbon bonds in one or more fatty acids in the lipid;substituting —Si(CH₃)₂- or —Si(—CH₂—(CH₂)_(n)—CH₂-)- for one or more ofthe cis-unsaturated carbon-carbon bonds to produce a silyl lipid; andproducing an improved preparation of lipid nanoparticles that containthe silyl lipid. Any of the lipids shown in FIGS. 3A and 3B can beconverted to silyl counterparts in this fashion.

Characterization of Silyl-Lipid Containing LNPs

The biophysical properties of novel silyl LNPs are measured for liposomeformation and silyl-LNP formulation and established throughcharacterization techniques including measurements of zeta potential,transmission electron microscopy and small-angle X-ray scattering. TheRNA/silyl-LNP complex is characterized using HPLC to quantify RNAencapsulation. In cellulo studies can compare transfection efficacy ofsiRNA and cytotoxicity of silyl-lipid LNPs with commercially availableliposome RNA delivery systems.

Pharmaceutical Payloads and Therapeutic Applications

Silyl lipid containing lipid nanoparticles according to this disclosurecan be used for delivering any pharmaceutical payload in vivo that theclinician or investigative scientist may wish to use. Suitable are smallmolecule pharmaceutical agents (less than 100 Da), proteins, and nucleicacids of various kinds, or a combination thereof. Treatment is done byadministering to a subject an amount of the agent-carrying LNPs that iseffective in achieving one or more clinical aims. Packaging thepharmaceutical agent in a silyl LNP may enhance stability of the agentor composition during storage, and/or help promote endocytosis or otherentry of the pharmaceutical agent into a target cell in a treatedsubject.

Silyl LNPs are particularly advantageous for delivering a nucleic acidor mixture thereof for purposes of immunization or gene therapy.Illustrative payloads for immunogenic compositions or vaccines are shownin TABLE 2, exemplified by mRNA vaccines. The nucleic acid encodes oneor more epitopes from the intended immune target, and optionally one ormore proteins that may act as an adjuvant or stimulant to enhanceimmunogenicity. The target may be an infectious agent, such as apathogenic virus, bacteria, or protozoan. Alternatively, the target maybe a cancer cell, in which case the encoded epitopes are epitopesexpressed by the cancer cell that are specific to the cancer or to thetissue type.

For example, the technology of this disclosure can be used to prepare acomposition to induce a response to the SARS-CoV-2 virus, for thepurpose of prevention or treatment of COVID-19. Representativeimmunogenic epitopes may be taken from any one or more of the fourSARS-CoV-2 structural proteins: namely, membrane glycoprotein (M),envelope protein (E), nucleocapsid protein (N), and the spike protein(S). Most current vaccines against SARS-CoV-2 typically include orencode the whole spike protein. Ways to optimize the spike protein wererecently discussed by F. Heinz & K. Stiasny, NPJ Vaccines (2021) 6:104.

TABLE 2 LNP-mRNA immunogenic compositions Clinical Trials Name DiseaseEncoded antigen identifier Phase Infections mRNA-1273 SARS-CoV-2 SpikeNCT04470427 III (EUA and CMA) BNT162b2 SARS-CoV-2 Spike NCT04368728 III(EUA and CMA) CVnCoV SARS-CoV-2 Spike NCT04652102 III LNP- SARS-CoV-2Spike ISRCTN17072692 I nCoVsaRNA ARCT-021 SARS-CoV-2 Spike NCT04728347II ARCoV SARS-CoV-2 Receptor-binding ChiCTR2000034112 I domain mRNA-1440Influenza H10N8 Haemagglutinin NCT03076385 I mRNA-1851 Influenza H7N9Haemagglutinin NCT03345043 I mRNA-1893 Zika virus Pre-membrane andNCT04064905 I envelope glycoproteins mRNA-1345 Respiratory FglycoproteinNCT04528719 I syncytial virus mRNA-1653 Metapneumovirus MPV and PIV3 FNCT03392389 I and parainfluenza glycoproteins virus type 3 (MPV/PIV3)mRNA-1647 Cytomegalovirus Pentameric complex NCT04232280 II and Bglycoprotein mRNA-1388 Chikungunya Chikungunya virus NCT03325075 I virusantigens CV7202 Rabies virus G glycoprotein NCT03713086 I CancermRNA-5671/ Non-small-cell KRAS antigens NCT03948763 I V941 lung cancer,colorectal cancer, pancreatic adenocarcinoma mRNA-4157 MelanomaPersonalized NCT03897881 II neoantigens mRNA-4650 GastrointestinalPersonalized NCT03480152 I/II cancer neoantigens FixVac MelanomaNY-ESO-1, tyrosinase, NCT02410733 I MAGE-A3, TPTE TNBC-MERITTriple-negative Personalized NCT02316457 I breast cancer neoantigensHARE-40 HPV-positive HPV oncoproteins NCT03418480 I/II cancers E6 and E7RO7198457 Melanoma Personalized NCT03815058 II neoantigens W_ova1Ovarian cancer Ovarian cancer NCT04163094 I antigens

The technology of this disclosure can also be used for the purpose ofgene therapy, which is the delivery of a nucleic acid to a subject forthe purpose of therapy. Therapeutic purposes include but are not limitedto expression of a therapeutic protein encoded in the nucleic acid (suchas a cytokine or anti-cancer agent), expression of an essential proteinthat the subject is unable to produce themselves, delivery of a geneediting system such as CRISPR/Cas9 or a guide RNA; or immunization ofthe subject against a pathogen or antigen encoded in the nucleic acid.Other payloads that can be used for gene therapy include DNA antisenseoligonucleotides, DNA aptamers; micro RNAs, short interfering RNAs,ribozymes, RNA decoys and circular RNAs that specifically increase ordecrease expression of a particular endogenous gene in the subject or aninfectious agent. K. Sridharan et al., Br J Clin Pharmacol. 2016 Sep;82(3): 659-672.

Illustrative payloads for gene therapy are shown in TABLE 3. In theexamples shown, the nucleic acid encodes a therapeutic antibody (forpassive immunization), anti-cancer drugs such as cytokines andchemotactic factors (for cancer treatment), and natural human proteins(to promote synthesis of an essential factor that the subject may belacking, such as in the case of a genetically inherited condition).TABLES 2 and 3 are adapted from X. Hou et al., Lipid nanoparticles formRNA delivery, Nat Rev Materials 2021, 10:1-17.

TABLE 3 LNP pharmaceuticals for gene therapy Clinical Trials NameDisease Encoded protein identifier Phase Infections mRNA-1944Chikungunya virus Antibody against NCT03829384 I chikungunya virusCancer mRNA 2416 Solid tumors OX40L NCT03323398 II mRNA-2752 Solidtumors OX40L, IL-23 and IL-36γ NCT03739931 I MEDI1191 Solid tumors IL-12NCT03946800 I SAR441000 Solid tumors IL-12sc, IL-15sushi, NCT03871348 IIFNα and GM-CSF Genetic disorders mRNA-3704 MethylmalonicMethylmalonyl-CoA NCT03810690 I/II acidaemia mutase mRNA-3927 PropionicPropionyl-CoA NCT04159103 I/II acidaemia carboxylase MRT5201 OrnithineOrnithine NCT03767270 I/II transcarbamylase transcarbamylase deficiencyMRT5005 Cystic fibrosis Cystic fibrosis NCT03375047 I/II transmembraneconductance regulator NTLA-2001 Transthyretin CRISPR-Cas9 geneNCT04601051 I amyloidosis with editing system polyneuropathy

Medicaments and Commercial Products

Preparation and formulation of pharmaceutical agents for use accordingto this disclosure can incorporate standard technology, as described,for example, in the most recent edition of Remington: The Science andPractice of Pharmacy. The formulation will typically be optimized foradministration systemically, either intramuscularly or subcutaneously,or for administration orally or nasally (for example, to stimulate themucosal immune system).

Silyl LNP preparations may be provided as one or more unit doses (eithercombined or separate), each containing an amount of the pharmaceuticalpayload in LNP form that is effective in the treatment of a chosendisease, infection, or clinical condition. The commercial product maycontain a device such as a syringe for administration of the agent orcomposition in or around the target tissue of a subject in need thereof.The product may also contain or be accompanied by an informationalpackage insert describing the use and attendant benefits of the LNPs intreating the condition for which it is indicated and approved.

This disclosure also includes kits of chemical compounds, theirpreparation and use. Such kits may contain a combination of “buildingblocks” that are useful in constructing or fine-tuning silyl lipids,such compounds selected from what is shown in FIG. 2 . Other kits mayinclude preparations of silyl lipids (optionally in combination withemulsion agents) for use in encapsulating a drug or vaccine payload intoa silyl LNP preparation as put forth herein.

Terms Used in this Disclosure

The term “lipid” as used in this disclosure refers generally to anorganic amphipathic molecule having a charged or ionizable headgroup,and one or more lipophilic tails. The tails are typically linear orbranched hydrocarbons, which may be either saturated or unsaturated. Thetails may be referred to informally as fatty acids, but this places noconstraint on the linkage of the lipophilic tails to the headgroup.

A “lipid nanoparticle” (LNP) is defined as any lipid-containing particleof microscopic or sub-microscopic size, typically but not necessarilyless than 1000 nanometers in diameter. LNPs developed for delivery of atherapeutic payload may be (1) a lipophilic or amphipathic solid orliquid core coated with a single lipid layer, (2) a liposome or otherparticle comprising a lipid bilayer and a solid, liquid, or bufferedinterior, or (3) a multilamellar structure. The “payload” contains oneor more pharmaceutically active compounds (or a placebo equivalentthereof), optionally combined with one or more pharmaceuticallyacceptable excipients, packing agents, or other compounds.

A “silyl” or “silylated” lipid is a molecule that contains a polarheadgroup and one or more alkyl or fatty acid tails. At least one of thetails contains one or more silicon atoms that interconnects portions ofthe tail. A “silyl LNP” is an LNP in which at least some of the lipids(typically 5% or more wt/wt) are silyl lipids.

In the context of this disclosure, the term “alkyl,” by itself or aspart of another substituent refers to a straight or branched aliphaticradical having the number of carbon atoms indicated. The term “alkoxy,”by itself or as part of another substituent, refers to a group havingthe formula —OR, wherein R is alkyl. A carbonyl is a C═O group that isdivalent at the carbon atom, and double-bonded to an oxygen. The term“alkylene” refers to an alkyl group containing an alkene linking atleast two other alkyl groups moieties. The two moieties linked to thealkylene group may be linked to the same carbon atom or different carbonatoms of the alkylene group. The term “alkene” refers to a straight orbranched, divalent carbon chain having one or more carbon-carbon doublebonds (—CR═CR′—, wherein R and R′ are each independently hydrogen or afurther substituent). A “cis alkene” contains the structure —CH═CH′—wherein the H atoms are in the cis position. A “heteroalkyl” group is analkyl group in which one or more of the constituent carbon atoms havebeen replaced by nitrogen, oxygen, or sulfur. More generally, a“hydrocarbon” (unless otherwise specified) may be linear or branched,saturated or unsaturated, and may contain cycles or aromatic group. Asubstituted hydrocarbon contains additional atoms that are not carbon orhydrogen in any stable position. Unless otherwise stated or required,other chemical terms have their ordinary meaning.

Unless otherwise stated or required, each of the compound structuresreferred to in the disclosure include conjugate acids and bases havingthe same structure, crystalline and amorphous forms of those compounds,pharmaceutically acceptable salts, and prodrugs. This includes, forexample, tautomers, polymorphs, solvates, hydrates, unsolvatedpolymorphs (including anhydrates). For compounds having one or morechiral centers, if an absolute stereochemistry is not expresslyindicated, then each center may independently be of R-configuration orS-configuration or a mixture thereof.

A “vaccine” or “immunogenic compound” is a pharmaceutical preparationformulated to initiate an immune response (both B and T lymphocytes)that is specific for a pre-determined antigen or pathogen. It willtypically contain or encode one or more epitopes of the antigen orpathogen, packaged in a formulation that is effective in delivering theepitopes to antigen-presenting cells in a subject to which it isadministered, which in turn prompts the immune system of the subject toinitiate or boost the intended immune response. “Vaccination” or“immunization” is the act of administering such a pharmaceuticalcomposition to a person in need thereof, whether or not they have aparticular disease or condition. “Gene therapy” means administering anucleic acid to a subject in need thereof for the purpose of deliveringthe nucleic acid in a functional form to a target cell or tissue in thesubject for a therapeutic purpose, as described above.

A “therapeutically effective amount” is an amount of a compound of thepresent disclosure that (i) treats the particular disease, condition, ordisorder, (ii) attenuates, ameliorates, or eliminates one or moresymptoms of the particular disease, condition, or disorder, (iii)prevents or delays the onset of one or more symptoms of the particulardisease, condition, or disorder described herein, (iv) prevents ordelays progression of the particular disease, condition or disorder, or(v) at least partially reverses damage caused by the condition prior totreatment.

Successful “treatment” of a condition according to this disclosure mayhave any effect that is beneficial to the subject being treated. Thisincludes decreasing severity, duration, or progression of a condition,or of any adverse signs or symptoms resulting therefrom. Treatment mayalso be unsuccessful, resulting in no improvement in typical signs andsymptoms of the condition. The subject has still been “treated” if theintention of the managing clinician has been at least in part theimprovement or alteration of a condition referred to. A concurrentobjective of therapy is to minimize adverse effects on the target tissueor elsewhere in the treated subject.

Published Information

The reader may be guided in their practice of the technology disclosedhere by referring to general information and techniques in the followingreference texts:

-   -   Solid Lipid Nanoparticles: a Nano Carrier for Drug Delivery by        Sukanta Satapathy, Chandra Sekhar Patro, et al., 2021    -   Solid Lipid Nanoparticle: A compendious approaches in industrial        scale-up techniques of Solid lipid Nanoparticles and Biomedical        applications by Deep M. K. and Karthikeyan M,2020    -   Liposomes: Drug and Gene Delivery Systems (Biomaterials Science)        by Arabinda Chaudhuri, 2022    -   The Chemistry of Organic Silicon Compounds, by Zvi Rappoport and        Yitzhak Apeloig, 2001    -   Gene Transfer, Gene Therapy And Genetic Pharmacology by Daniel        Scherman, 2019    -   Design and Development of Novel Drugs and Vaccine by Tarun Kumar        Bhatt and Surendra Nimesh, 2021

Also potentially of interest are the general information and techniquespresented in the following published articles:

-   -   Hou X, Zaks T, Langer R, Dong Y. Lipid nanoparticles for mRNA        delivery. Nat Rev Materials 2021, 10:1-17.    -   Schoenmaker, L.; Witzigmann, D.; Kulkarni, J. A.; Verbeke, R.;        Kersten, G.; Jiskoot, W.; Crommelin, D. J. A. MRNA-Lipid        Nanoparticle COVID-19 Vaccines: Structure and Stability. Int. J.        Pharm. 2021, 601 (March), 120586.    -   Arpicco, S.; Canevari, S.; Ceruti, M.; Galmozzi, E.; Rocco, F.;        Cattel, L. Synthesis, Characterization and Transfection Activity        of New Saturated and Unsaturated Cationic Lipids. Farm. 2004, 59        (11), 869-878.    -   Ferrari, M. E.; Rusalov, D.; Enas, J.; Wheeler, C. J. Synergy        between Cationic Lipid and Co-Lipid Determines the Macroscopic        Structure and Transfection Activity of Lipoplexes. Nucleic Acids        Res. 2002, 30 (8), 1808-1816.    -   Heyes, J.; Palmer, L.; Bremner, K.; MacLachlan, I. Cationic        Lipid Saturation Influences Intracellular Delivery of        Encapsulated Nucleic Acids. J. Control. Release 2005, 107 (2),        276-287.    -   Zhi, D.; Zhang, S.; Wang, B.; Zhao, Y.; Yang, B.; Yu, S.        Transfection Efficiency of Cationic Lipids with Different        Hydrophobic Domains in Gene Delivery. Bioconjug. Chem. 2010, 21        (4), 563-577.    -   Zhang, Y.; Sun, C.; Wang, C.; Jankovic, K. E.; Dong, Y. Lipids        and Lipid Derivatives for RNA Delivery. Chem. Rev. 2021.    -   Showell, G. A.; Mills, J. S. Chemistry Challenges in Lead        Optimization: Silicon Isosteres in Drug Discovery. Drug Discov.        Today 2003, 8 (12), 551-556.    -   Ramesh, R.; Reddy, D. S. Quest for Novel Chemical Entities        through Incorporation of Silicon in Drug Scaffolds. J. Med.        Chem. 2018, 61 (9), 3779-3798.    -   Bains, W.; Tacke, R. Silicon Chemistry as a Novel Source of        Chemical Diversity in Drug Design. Curr. Opin. Drug Discov.        Devel. 2003, 6, 526-543.    -   Zakai, U. I.; Bikzhanova, G.; Staveness, D.; Gately, S.;        West, R. Synthesis of Lipophilic Sila Derivatives of        N-Acetylcysteineamide, a Cell Permeating Thiol. Appl. Organomet.        Chem. 2010, 24 (3), 189-192.    -   Gately, S.; West, R. Novel Therapeutics with Enhanced Biological        Activity Generated by the Strategic Introduction of Silicon        Isosteres into Known Drug Scaffolds. Drug Dev. Res. 2007, 68,        156-163.    -   Nakamura, M.; Kajita, D.; Matsumoto, Y.; Hashimoto, Y. Design        and Synthesis of Silicon-Containing Tubulin Polymerization        Inhibitors: Replacement of the Ethylene Moiety of Combretastatin        A-4 with a Silicon Linker. Bioorg. Med. Chem. 2013, 21 (23),        7381-7391.    -   Blakney, A. K.; McKay, P. F.; Yus, B. I.; Aldon, Y.;        Shattock, R. J. Inside out: Optimization of Lipid Nanoparticle        Formulations for Exterior Complexation and in vivo Delivery of        SaRNA. Gene Ther. 2019, 26 (9), 363-372.    -   Fletcher, S.; Ahmad, A.; Perouzel, E.; Heron, A.; Miller, A. D.;        Jorgensen, M. R. In vivo Studies of Dialkynoyl Analogues of        DOTAP Demonstrate Improved Gene Transfer Efficiency of Cationic        Liposomes in Mouse Lung. J. Med. Chem. 2006, 49 (1), 349-357.    -   Semple, S. C.; Akinc, A.; Chen, J.; Sandhu, A. P.; Mui, B. L.;        Cho, C. K.; Sah, D. W. Y.; Stebbing, D.; Crosley, E. J.;        Yaworski, E. Rational Design of Cationic Lipids for SiRNA        Delivery. Nat. Biotechnol. 2010, 28 (2), 172-176.    -   Akinc, A.; Zumbuehl, A.; Goldberg, M.; Leshchiner, E. S.;        Busini, V.; Hossain, N.; Bacallado, S. A.; Nguyen, D. N.; A        combinatorial approach to determine functional group effects on        lipidoid-mediated siRNA delivery. Bioconjug Chem. 2010 Aug. 18;        21(8): 1448-1454.    -   Rietwyk, S. and Peer, D. Next Generation Lipids in RNA        Interference Therapeutics. ACS Nano 2017, 11, 7572-7586.    -   Han, X.; Zhang, H.; Butowska, K.; Swingle, K.; Alameh, M.;        Weissman, D.; Mitchell, M. An Ionizable Lipid Toolbox for RNA        Delivery. Nature Communications 2021, 12, 7233.    -   Hou, X.; Zaks, T.; Langer, R.; Dong, Y. Lipid Nanoparticles for        mRNA Delivery. Nature Reviews Materials 2021, 6, 1078-1094.    -   Li, S. and Huang, L. Pharmacokinetics and Biodistribution of        Nanoparticles. Mol. Pharmaceutics 2008, 5, 4, 496-504.

Incorporation by Reference

For all purposes, each and every publication and patent document citedin this disclosure is hereby incorporated herein by reference in itsentirety for all purposes to the same extent as if each such publicationor document was specifically and individually indicated to beincorporated herein by reference. This includes the information providedin X. Hou et al., Lipid nanoparticles for mRNA delivery, Nat RevMaterials 2021, 10:1-17, and U.S. Pat. Nos. 9,393,215 and 10,266,485.

Practice of the Invention

The technology provided in this disclosure and its use is describedwithin a hypothetical understanding of general principles of lipidchemistry, nucleic acid chemistry, and the formulation and use ofpharmaceutical products. These discussions are provided for theedification and interest of the reader, and are not intended to limitthe practice of the claimed invention. The silyl lipids of thedisclosure can be used for various purposes, including but not limitedto the making lipid nanoparticles. In turn the LNPs of the disclosurecan be used for various purposes, including but not limited a role as adelivery vehicle for pharmaceutical agents. All of the products andmethods claimed in this application may be used for any suitable purposewithout restriction, unless otherwise indicated or required. The silyllipids and LNPs are believed to be safe, but at the time of thiswriting, have not been tested for safety or efficacy in human subjects.

While the invention has been described with reference to the specificexamples and illustrations, changes can be made and equivalents can besubstituted to adapt the technology to a particular context or intendeduse as a matter of routine development and optimization and within thepurview of one of ordinary skill in the art, thereby achieving benefitsof the invention without departing from the scope of what is claimed andtheir equivalents.

The invention claimed is:
 1. An immunogenic composition formulated foradministration to a subject for prevention or treatment of COVID-19,comprising a lipid nanoparticle that contains a drug payload; wherein atleast 2% of lipid molecules in the nanoparticles are silyl lipids havingthe structure shown in Formula III or Formula IIIa:

wherein R¹, R², and R³ are independently H, —CH₃, or a substituted orunsubstituted hydrocarbon containing two to ten carbon atoms, X¹ isnitrogen or —(OPO₃)—, X² and X³ are independently methylene or carbonylor together are a quaternary carbon; R⁴ and R⁵ are independentlyselected from methyl and a hydrocarbon or substituted hydrocarbon of 4to 12 carbon atoms; R^(8a) and R^(8b) are independently methyl or asaturated or unsaturated hydrocarbon of 2 to 10 carbon atoms; n=1 to 4;n1=1 to 6; n2, n3, n4 and n5 are independently 4 to 12; and wherein thedrug payload includes one or more protein components of SARS-CoV-2 (thevirus that causes COVID-19) and/or one or more nucleic acids encodingsuch protein components.
 2. The immunogenic composition of claim 1,wherein the drug payload comprises a messenger RNA (mRNA) encoding allor at least an immunogenic portion of the spike protein of SARS-CoV-2.3. A preparation of lipid nanoparticles, in which at least 5% of lipidsin the nanoparticles are silyl lipids having the structure shown inFormula III or Formula IIIa:

wherein R¹, R², and R³ are independently H, —CH₃, or a substituted orunsubstituted hydrocarbon containing two to ten carbon atoms, X¹ isnitrogen or —(OPO₃)—, X² and X³ are independently methylene or carbonylor together are a quaternary carbon; R⁴ and R⁵ are independentlyselected from methyl and a hydrocarbon or substituted hydrocarbon of 4to 12 carbon atoms; R^(8a) and R^(8b) are independently methyl or asaturated or unsaturated hydrocarbon of 2 to 10 carbon atoms; n=1 to 4;n1=1 to 6; n2, n3, n4 and n5 are independently 4 to
 12. 4. Thecomposition of claim 3, wherein the nanoparticles contain a drug payloadwhich is a nucleic acid.
 5. A silyl lipid having the structure shown inFormula III or Formula IIIa:

wherein R¹, R², and R³ are independently H, —CH₃, or a substituted orunsubstituted hydrocarbon containing two to ten carbon atoms, X¹ isnitrogen or —(OPO₃)—, X² and X³ are independently methylene or carbonylor together are a quaternary carbon; R⁴ and R⁵ are independentlyselected from methyl and a hydrocarbon or substituted hydrocarbon of 4to 12 carbon atoms; R^(8a) and R^(8b) are independently methyl or asaturated or unsaturated hydrocarbon of 2 to 10 carbon atoms; n=1 to 4;n1=1 to 6; n2, n3, n4 and n5 are independently 4 to
 12. 6. Thecomposition of claim 1, wherein X¹ is nitrogen.
 7. The composition ofclaim 1, wherein n1 is 1, R¹ and R² are both —H or —CH₃, and R³ is ahydrocarbon containing two to ten carbon atoms.
 8. The composition ofclaim 1, wherein X² and X³ are both methylene or both carbonyl.
 9. Thecomposition of claim 1, wherein n2, n3, n4, and n5 are all
 7. 10. Thecomposition of claim 1, wherein R⁴ and R⁵ in Formula III are both methyland n in Formula IIIa is
 1. 11. The composition of claim 1, whereinR^(8a) and R^(8b) are both methyl.
 12. The composition of claim 1,wherein the nanoparticles are liposomes.
 13. The composition of claim 1,wherein at least 25% of lipids in the nanoparticles are silyl lipids.14. The composition of claim 1, wherein at least 50% of the silyl lipidsin the nanoparticles are cationic or comprise a headgroup that isionizable.
 15. The composition of claim 1, wherein at least 5% of thesilyl lipids in the nanoparticles are PEGylated, whereby any one or moreof R¹, R², and R³ contain —O—[CH₂—CH₂—O- ]_(n9)-R⁹, wherein R⁹ is —OH,—OCH₃, —NH₂, or a substituted hydrocarbon, and n9 is an integer of 10 ormore.
 16. The composition of claim 1, wherein the lipid nanoparticlescomprise a proportion of cationic lipids, ionizable lipids, PEGylatedlipids, and optionally cholesterol.
 17. The composition of claim 1,wherein the lipid nanoparticle contains any one or more of the followingcomponents in silylated form: DOTMA(1,2-di-O-octadecenyl-3-trimethylammonium-propane), DOTAP(N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride), DOPE(2-dioleoyl-sn-glycero-3-phosphoethanolamine), and PEGylated DOPE. 18.The composition of claim 1, wherein the solid nanoparticles or liposomeshave an median diameter between 20 and 500 nm.
 19. A method of inducinga specific immune response in a subject in need thereof, comprisingadministering to the subject a composition according to claim
 1. 20. Amethod of reducing risk of a subject of contracting COVID-19, the methodcomprising administering to the subject a composition according to claim1.